Downregulation of microRNA‐124‐3p promotes subventricular zone neural stem cell activation by enhancing the function of BDNF downstream pathways after traumatic brain injury in adult rats

Abstract Aims In this study, the effect of intracerebral ventricle injection with a miR‐124‐3p agomir or antagomir on prognosis and on subventricular zone (SVZ) neural stem cells (NSCs) in adult rats with moderate traumatic brain injury (TBI) was investigated. Methods Model rats with moderate controlled cortical impact (CCI) were established and verified as described previously. The dynamic changes in miR‐124‐3p and the status of NSCs in the SVZ were analyzed. To evaluate the effect of lateral ventricle injection with miR‐124‐3p analogs and inhibitors after TBI, modified neurological severity scores (mNSSs) and rotarod tests were used to assess motor function prognosis. The variation in SVZ NSC marker expression was also explored. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of predicted miR‐124‐3p targets was performed to infer miR‐124‐3p functions, and miR‐124‐3p effects on pivotal predicted targets were further explored. Results Administration of miR‐124 inhibitors enhanced SVZ NSC proliferation and improved the motor function of TBI rats. Functional analysis of miR‐124 targets revealed high correlations between miR‐124 and neurotrophin signaling pathways, especially the TrkB downstream pathway. PI3K, Akt3, and Ras were found to be crucial miR‐124 targets and to be involved in most predicted functional pathways. Interference with miR‐124 expression in the lateral ventricle affected the PI3K/Akt3 and Ras pathways in the SVZ, and miR‐124 inhibitors intensified the potency of brain‐derived neurotrophic factor (BDNF) in SVZ NSC proliferation after TBI. Conclusion Disrupting miR‐124 expression through lateral ventricle injection has beneficial effects on neuroregeneration and TBI prognosis. Moreover, the combined use of BDNF and miR‐124 inhibitors might lead to better outcomes in TBI than BDNF treatment alone.


| INTRODUC TI ON
Traumatic brain injury (TBI) is a leading cause of injury-related death and disability worldwide and has a devastating impact on patients and their families. 1 TBI is characterized by a heterogeneous group of pathologies initiated by diverse mechanisms and disruptions to various neurological functions, such as executive actions, cognitive ability, attention, memory data processing, and language abilities. 2 For years, updates to treatment guidelines based on strong evidence for TBI treatment have been slow, and many multicenter clinical trials of medical interventions that have promising preclinical results not led to significant differences in neurological outcomes. 1,3 The adult mammalian brain is capable of structural and functional repair, plasticity, and regeneration. Adult neural stem cells (NSCs) residing in the subventricular zone (SVZ) produce DCX + neuroblasts that migrate along the rostral migratory stream and differentiate into neurons. 4 NSCs need to exit the quiescence state to undergo neurogenesis and differentiation. This process is controlled in vivo by comprehensive signaling processes involving, for example, local cellcell interactions, secreted factors in the blood or cerebrospinal fluid, and long-range signaling triggered by neuronal inputs or factors secreted from the choroid plexus in the SVZ niche. 5 After brain injury, the environment changes in the SVZ stem cell niche; for example, vasculature permeability is increased, inducing NSCs to migrate to the injured cortex and differentiate into protective astrocytes. 6 Additionally, DCX + neuroblasts continuously migrate to the corpus callosum 2-4 days after brain injury. 7 Transcriptome analysis based on single-cell RNA sequencing (RNA-seq) has revealed an increase in the proportion of "primed" quiescent and active NSCs in the SVZ 2 days post-ischemia. Both primed and active NSCs drive the expression of genes related to protein synthesis and the cell cycle. 8 These findings have highlighted reactive SVZ NSCs conferred protection after TBI, and therefore, promoting SVZ NSC activation might provide beneficial outcomes for patients.
MicroRNAs (miRNAs) are single-stranded noncoding RNAs 18-25 (~22) nucleotides in length that play important roles in the posttranscriptional regulation of gene expression. 9 To date, 2588 mature miRNAs processed from 1881 precursor miRNAs have been annotated in the human genome, 10 and most of these miRNAs are highly conserved across species. 11 miRNAs regulate many target genes through both perfect and imperfect base pairing of the miRNA seed region to the 3′ untranslated region (UTR) of mRNA, making it difficult to gain a complete understanding of the complex miRNA function. 12 MiR-124 is one of the most abundant miRNAs expressed in the adult brain and has been found to be involved in both prenatal and postnatal neuronal differentiation. 13 MiR-124 regulates Sox9 to impair NSC proliferation and facilitate NSC differentiation. 14 In the case of cerebral ischemia, the level of miR-124 is reduced in the SVZ, and this reduction inhibits neuronal progenitor cell proliferation because jagged-1 (JAG1) expression is repressed. 15 An in vitro study of oxygen-glucose deprivation/re-oxygenation (OGD/R) showed that a miR-124 inhibitor promoted growth-associated protein-43 (GAP-43) expression and axonal growth. 16 Although the function of miR-124 in cerebral ischemia and other central nervous system diseases has been previously and extensively explored, 17 the role that miR-124 plays in SVZ NSCs in vivo after TBI remains unclear.
In the present study, we used an in vivo controlled cortical impact (CCI) rat model to explore the function of miR-124 in adult SVZ NSCs after TBI. We found that lateral cerebral ventricle injection with miR-124 inhibitors enhanced SVZ NSC proliferation and improved motor function in adult TBI rats. Based on the results of a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on predicted miR-124 targets, we verified that this regulation pattern was partially implemented and affected the function of pathways downstream of BDNF, such as the PI3K/Akt3 and Ras signaling pathways. Medical University. Rats were raised in the facility with a 12-h day and night cycle and with stable temperature and humidity. All rats were allowed to eat and drink freely and had adequate space for free activity. A CCI device (Hatteras Instruments Inc., Cary, NC, USA) was used to establish the TBI model. After anesthetization with 10% chloral hydrate (intraperitoneal injection), the rats were placed on a brain stereotaxis instrument (Kopf Instruments, Tujunga, CA, USA).

| Controlled cortical impact (CCI) model
The rat head was wiped with 70% ethanol, and the scalp was cut at the midline. A bone harvest drill was used to make a hole 2.5 mm in diameter, 2.0 mm posterior to the bregma, and 2.0 mm lateral to the sagittal suture on the right side of the skull. The CCI device was then used to induce a moderate TBI model with a general parameter use of BDNF and miR-124 inhibitors might lead to better outcomes in TBI than BDNF treatment alone.

K E Y W O R D S
bioinformatics, brain-derived neurotrophic factor (BNDF), miR-124, neural stem cells (NSCs), subventricular zone (SVZ), traumatic brain injury (TBI) (impact depth, 3 mm; impact velocity, 5.0 m/s; dwell time, 100 ms). 18 After the injury, the scalp incision was closed, and the rats were placed on a constant temperature plate to maintain their core temperature. The research strategy used in this study complied with the instructions of the Animal Research Reporting in vivo Experiment (ARRIVE) 2.0 guidelines. 19

| Administration of drugs
Rats were anesthetized with 10% chloral hydrate (intraperitoneal injection) and placed in a stereotaxic frame. Stereotaxic injections were made with a Hamilton syringe using the following coordinates:  with NC, agomir, or antagomir RNA and administered by bilateral intraventricular injection to each rat. After injection, the needle was left in place for 10 min before being slowly withdrawn. The mortality rate of the rats after surgery was 1/7, and no difference was found among the different treatment groups. The cause of death of rats that died 24 h after surgery was ascribed to anesthesia or stress, and these rats were excluded from the functional analysis.

| Neurobehavioral training and evaluation
The mNSS was obtained prior to TBI (or after sham surgery) and at 1, 3, 5, 7, 14, and 30 days after TBI (or sham) surgery. Briefly, the test included a composite of tasks that the rats completed for the assessment of the motor, sensory, reflex, and balance ability postinjury. Rats with an abnormal score (score > 0) before surgery were excluded from the experiment. 21 The higher the score was, the more serious the defect was considered.
A rotarod test was performed as described herein. 22,23 Briefly, rats were placed on a rotating drum with a speed slowly increasing from 0 to 40 rpm within 5 min; that is, the speed increased 2 rpm every 10 s for 200 s. When the speed was held constant at 40 rpm, the duration (seconds) that the rat stayed on the rod was recorded; that is, the time between the initiation of the test and the time at which the animal fell off the drum was recorded. Animals were tested three times per day for 3 days before surgery.

| Tissue preparation
Rats with only CCI were sacrificed 1, 3, 5, and 7 days after the operation. The rats in the injection group were sacrificed 7 days after CCI induction. Rats were deeply anesthetized and transcardially per-

| qRT-PCR assay
Total RNA was isolated from SVZ tissue using TRIzol reagent

| Western blotting
The total protein in the SVZ tissue was extracted, and a BCA protein kit (Beyotime) was used to quantify the protein concentrations. Ten  Table 1.

| Statistical analysis
All of the data are expressed as the means ± SD. GraphPad Prism 7.0 software was used to perform Student's t-test or two-way ANOVA followed by Tukey's multiple comparison test. Nonparametric tests (Jonckheere-Terpstra test and Kruskal-Wallis test) were performed with data that were not normally distributed. p < 0.05 was considered to be significant.

| Time course of miR-124-3p and SVZ NSCs post-TBI
The expression of miR-124-3p in the SVZ was investigated 1, 3, 5, 7, 14, and 30 days post-TBI (n = 3 per group), and qRT-PCR showed a downregulation of miR-124-3p. The expression of miR-124-3p decreased dramatically in the first 3 days post-TBI, and then, the expression level increased slightly but remained lower than the physiological level and was sustained at this level until day 30 ( Figure 1A). The status of the NSCs in the SVZ was also explored by immunostaining 3, 5, and 7 days post-TBI (n = 6 per group) ( Figure 1B,C). The results showed that the number of GFAP + / Nestin + stem cells and DCX + progenitor cells increased gradually after TBI ( Figure 1D,E). The number of Ki67 + dividing cells increased and reached a peak on day 5 ( Figure 1F). These data suggested that

| MiR-124-3p increased neurological deficits after TBI
To confirm the role of miR-124-3p after TBI, we administered miR-124-3p to the CCI rats. As SVZ NSCs extend cilia through the ependymal rosettes to contact the cerebrospinal fluid in the ventricular space, 26 we injected the lateral cerebral ventricle with miR-124-3p agomir or antagomir to explore their effects on TBI rats. qRT-PCR was used to explore the expression of miR-124 3 days after injection, and a dose of 400 pmol RNA was adopted as the appropriate intervention for the following experiments ( Figure 2A). Rats (n = 6 per group) were evaluated by mNSS and rotarod test, and the results showed no difference between the groups before surgery.
Following CCI and injection, the TBI rats exhibited worse mNSS or rotarod test performance than the sham rats ( Figure 2B,C). By consecutively monitoring the performance of each group for 30 days, no difference was found in mNSS between the TBI + NC group and the TBI + agomir group, but the TBI + antagomir group obtained better  Figure 2B). In the rotarod test, the TBI + antagomir group achieved the lowest fall ratio on days 5, 7, and 14, and the TBI + agomir group had the highest fall ratio ( Figure 2C). These results implied that downregulation of miR-124-3p correlated with a better motor function outcome in adult TBI rats.

| MiR-124-3p attenuates SVZ NSC activation post-TBI
To explore whether miR-124-3p exerted an impact on SVZ NSCs after TBI, we performed IF assays to explore the variation in NSC markers in each group on Day 7 post-TBI (n = 6 per group) ( Figure 3A,B).
Consistent with the behavioral outcomes described above, the TBI + antagomir group had the largest number of GFAP + /Nestin + cells, DCX + cells, and Ki67 + cells ( Figure 3C-E). For the TBI + NC group and TBI + agomir group, no difference was found in GFAP + /Nestin + cell numbers. The numbers of DCX + cells, and Ki67 + cells in the TBI + agomir group were lower than those in the TBI + NC group. The IF assay results indicated that decreased miR-124-3p might play a role in promoting NSC activation in the SVZ after TBI.

| Bioinformatics analysis of MiR-124-3p targets
The biological function of miR-124-3p was predicted through a bioinformatics analysis, the workflow of which is schematically presented in Figure 4A. Target genes of miR-124-3p were screened using TargetScan (http://www.targe tscan.org), miRDB (www.mirdb. org), and miRanda (www.miran da.org), and a total of 887 targets were screened for use in a pathway enrichment analysis. KEGG pathway analysis results based on the DAVID database ( Figure 4B) and Cytoscape ClueGO ( Figure 4C) showed that the predicted targets were mostly enriched in "neurotrophin signaling pathway," "prolactin signaling pathway" and "acute myeloid leukemia." Among the targets in these pathways, NRas, PIK3CA, and Akt3 were found to be participants in the most pathways. These results suggested that miR-124-3p broadly regulates these pathways and that NRas, PI3KCA, and Akt3 are the pivotal participants in these processes.
MiR-124-3p targets participated in Neutrophin signaling pathways and prolectin pathways are highlighted in Supplementary Materials.

| The MiR-124-3p antagomir promoted the expression of Akt3, PI3KCA, and Ras
We next detected the expression of Ras, PI3KCA, and Akt3 in all the experimental groups (n = 3 per group) 5 and 7 days after TBI ( Figure 5A,B). Western blot analysis showed that the expression of all three proteins increased after TBI, which indicated their proliferation-promoting effect after TBI. Additionally, the TBI + agomir group appeared to have lower levels of Akt3, PIK3CA, and Ras expression than the TBI + antagomir group, which implied that the expression of these genes was regulated by miR-124-3p. As the Ras, PI3K, and Akt are activated by phosphorylation, the expression of phospho-PI3K, phospho-MEK, and phospho-Erk was also tested.  , and 400 pmol per side, the injected RNAs all led to significant changes in miR-124-3p expression, and 400 pmol RNAs was applied in the following experiments. ⁎ p < 0.05, ⁎⁎ p < 0.01, and ⁎⁎⁎ p < 0.001 vs. the NC group. CCI rats then achieved double-sided lateral ventricle injection of NC, agomir or antagomir on the first day after injury and were then evaluated on the basis of mNSS scores (B) and a rotarod test (C). The results were analyzed by nonparametric test (mNSS) or ANOVA (rotarod), and the performance of the TBI + antagomir group was found to be better than that of the TBI + agomir or TBI + NC group. No significant difference was found between the TBI + agomir and TBI + NC groups. The data are presented as the means ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, and ⁎⁎⁎ p < 0.001 vs. the TBI + NC group. n = 6/group

| MiR-124-3p inhibited BDNF function in activating SVZ NSCs after TBI
To ascertain whether neurotrophins participate in the pathophysiological changes after TBI, we tested the expression of BDNF and NT-3 in the SVZ (n = 3 per group) from 1 to 7 days after surgery ( Figure 5C). We found that BDNF expression was upregulated gradually after TBI, while the expression of NT-3 was not altered. We also tested the expression of DCX, and in accordance with the expression of BDNF, the expression of DCX was found to have increased gradually after TBI. This finding suggested that BDNF participated in SVZ NSC activation. Then, we explored whether miR-124-3p di-

| DISCUSS ION
TBI, a vital cause of death and disability, causes untenable pain and places an unaffordable burden to both patients and their family members. 27 However, for decades, no drug has been sufficiently effective to alleviate the neurological disabilities caused by TBI. Adult mammalian NSCs, with self-renewal and differentiation capacity, ClueGO results based on the KEGG pathway analysis. Pathways with a p-value < 0.05 are visualized, and the relative target genes and their correlation between each pathway are shown. Pathways with the same color represent the same group whose gene overlap ratio was > 80% the effect of BDNF and further improve TBI prognosis is a promising approach. As BDNF is too large to permeate through the blood-brain barrier, 31 and therefore, direct BDNF injection into the CSF is recommended, as this provides additional beneficial effects to the NSCs along the lateral ventricles.
For the prolactin signaling pathway, for which the second highest score KEGG pathway analysis score was obtained, no method to evaluate its connection with TBI was discerned. Prolactin (PRL) is involved in a wide range of biological functions, including osmoregulation, lactation, reproduction, growth and development, endocrinology and metabolism, brain and behavior, and immunomodulation. 32 On the basis of the putative targets of miR-124, we identified 13 targets that were downstream of the prolactin signaling pathway, and these targets participated in the PI3K/Akt, MAPK, and Jak/STAT pathway. This result also offers clues to explain the reason that males and females have different outcomes after TBI. 35 One of the main limitations in our study is that we only used young adult male rats to assess the role of miR-124-3p in TBI prognosis and SVZ stem cell activation. Sex and age play important roles in determining the outcome of brain injury, 36,37 and premenopausal females often receive a better prognosis than males after brain injury, including TBI. 35 Individuals of different sexes suffer different pathophysiological changes after brain injury, including discrepant gene expression 38,39 and immunoreactions. 40 Therefore, investigation into the effects of miR-124-3p on rats of different sexes and ages separately is important, and future clinical trials should consider the impacts of sex and age on TBI prognosis in patient selection and classification.

| CON CLUS ION
In summary, our data indicated that downregulation of miR-124 after TBI enhanced SVZ NSC activation and further improved motor function in adult male rats. These effects were partially realized through intensification of pathway activation downstream of BDNF, such as the PI3K/Akt and Ras signaling pathways. Our exploration also indicated that bioinformatics analyses can lead to a comprehensive view for predicting miRNA functions and may be useful for providing more efficacious guidance for miRNA exploration.

ACK N OWLED G EM ENT
This work was supported by grants from the National Natural Science Foundation of China (grant 81971156) and Shanxi Science and Technology Foundation (2017JM8073).